Nonclassical monocytes (identified as CD14dimCD16+ in humans) exhibit a unique ability to `patrol' or survey the luminal side of the vascular endothelium both at steady state and during inflammation. Nonclassical monocytes function in circulation to aid in removing pathogens and debris from the vasculature. We recently found that nonclassical monocytes function in the vasculature to prevent tumor metastasis by orchestrating the killing and clearance of metastasizing tumor cells. The anti-tumor immune potential of nonclassical patrolling monocytes is in contrast to the growing evidence for pro-tumorigenic and pro-metastatic functions of myeloid cells in many tumor types. In the current proposal, we hypothesize that nonclassical monocytes function in an anti-tumoral manner to support CAR T cell expansion and efficacy in patients with solid tumors. Thus, one major goal of our proposal is to determine whether monocyte therapy using anti-tumoral nonclassical monocytes in combination with existing CAR T immunotherapy would improve efficacy. We will use mass cytometry to study monocyte subsets in cancer patients to identify new markers that will help readily determine how successful a proposed immunotherapy may be for patients. Aim 1 will identify unique markers of nonclassical monocytes in multiply relapsed sarcoma patients versus healthy subjects using mass cytometry. Aim 1 will be performed using banked samples from the existing GD2 CAR T trial at the NIH Clinical Center. Aim 2 will test the hypothesis that nonclassical monocytes play a functional role in promoting anti-tumor immunity when used in combination with immunotherapy for solid tumor metastasis. Aim 2 will use both xenografted humanized mouse and syngeneic mouse tumor models. Aim 3 will study the safety and preliminary efficacy of CAR T cell immunotherapy in combination with nonclassical monocyte cell therapy for patients with multiple relapsed, metastatic or progressive pediatric solid tumors. In Aim 3, we will initiate a second generation GD2 CAR T cell trial for multiply relapsed patients with osteosarcoma and neuroblastoma. Given the potential anti-tumor efficacy of patrolling monocytes we plan to initiate an early phase clinical trial delivering nonclassical monocytes alone and in combination with CAR T cell therapy as part of this trial. There are 3 investigators in this project: one extramural, one intramural at NCI, and one at the NIH Clinical Center. From the results of this proposal, we will achieve significant insight into the roles of various myeloid cell subpopulations on promoting and inhibiting the anti-tumoral responses of CAR T cell therapies for solid tumors. We are uniquely poised as a research team to directly address the role of monocytes and other myeloid cells in interacting with CAR T cells in human patients. The findings of our research team will significantly advance the knowledge that will lead to effective CAR T cell therapy for solid tumors. As similar interactions of monocytes occur with T cells that are modulated by checkpoint inhibitor trials, we anticipate that our findings will shed insight into monocyte:T cell interactions in the context of checkpoint inhibition therapy.